WO2006059877A1

WO2006059877A1 - Microorganism producing 5'-xanthylic acid and production method of 5'-xanthylic acid using the same

Info

Publication number: WO2006059877A1
Application number: PCT/KR2005/004080
Authority: WO
Inventors: Young Hoon Park; Hyoung Suk Kim; Jin Nam Lee; Ki Hoon Oh; Jeong Hwan Kim; Yoon Suk Oh; Jae Ick Sim; Kyung Oh Choi
Original assignee: Cj Corporation
Priority date: 2004-12-01
Filing date: 2005-12-01
Publication date: 2006-06-08
Also published as: TWI298743B; KR20060061135A; KR100588578B1; TW200634150A

Abstract

Disclosed is a mutant strain of Corynebacterium ammoniagenes CJXFT0301 (KCCM-10530) , Corynebacterium aimoniagenes CJXSul0401 (KCCM-10609) , which produces 5'- xanthylic acid in high concentrations and high yields by direct fermentation. Also disclosed is a method of producing 5 ' -xanthylic acid, which is based on directly accumulating 5 '-xanthylic acid in culture fluid in high concentrations and high yields using the mutant strain. The present microorganism shares the characteristics of the CJXFT0301 (KCCM-10530) strain, requirement for adenine and guanine, susceptibility to lysozymes and resistance to praline analogues, glutamine analogues and tryptophan analogues. Due to its sulfadiazine resistance, leading to increased synthesis of dihydropteroate and eventually elevated levels of tetrahydrofolic acid, the present microorganism directly accumulates 5 ' -xanthylic acid in culture fluid in high concentrations and high yields during the same fermentation duration.

Description

MICROORGANISM PRODUCING 5'-XANTHYLIC ACID AND PRODUCTION METHOD OF 5'-XANTHYLIC ACID USING THE SAME

Technical Field

The present invention relates to a microorganism producing 5'-xanthylic acid and a method of producing 5'- xanthylic acid using the microorganism. More particularly, the present invention relates to a mutant strain of Corynebacterium ammoniagenes CJXFT0301 (KCCM-10530) , Corynebacterium ammoniagenes CJXSul0401 (KCCM-10609) , which has sulfadiazine resistance, leading to increased synthesis of dihydropteroate and eventually elevated levels of tetrahydrofolic acid, thereby producing 5'-xanthylic acid in higher concentrations and higher yields than the CJXFT0301 (KCCM-10530) strain during the same fermentation duration by a direct fermentation technique, and a method of producing 5'-xanthylic acid using the mutant strain.

Background Art

5'-xanthylic acid, which is a metabolic intermediate in nucleotide biosynthesis, has physiological importance in animals and plants and is used in a wide spectrum of fields including food and pharmaceutical industries and for a variety of medical purposes. In particular, 5'-xanthylic acid is one of a group of nucleotides that are attractive as flavor-enhancing seasoning agents due to their high flavor-enhancing effects for food when used in combination with sodium glutamate. 5'-xanthylic acid is an intermediate in the biosynthesis of purine nucleotides and is important as a raw material for the production of 5'-guanylic acid. At present, microbial fermentation methods are widely used for producing 5'-guanylic acid having strong flavoring activity and high commercial value. With the microbial fermentation, it is most economical to produce 5'-xanthylic acid and enzymatically convert 5'-xanthylic acid to 5'-guanylic acid. Thus, 5'-xanthylic acid needs to be produced according to the demand for 5'-guanylic acid. Some methods are known for producing 5'-xanthylic acid, which include a method in which 5'-guanylic acid is produced by chemical synthesis or by degrading yeast ribonucleic acid and is deaminated; and a fermentative method in which a fermentation medium is supplemented with xanthine as a precursor, a microbial mutant is used, or the medium is supplemented with an antibiotic (Japanese Pat. Nos. Sho 42-1477 and Sho 44-20390) or a surfactant (Japanese Pat. Nos. Sho 42-3825 and Sho 42-3838).

As described above, many methods of producing 5'- xanthylic acid are known, and it is most important to economically produce high concentrations and high yields of 5'-xanthylic acid. Of the methods, a direct fermentative production of 5'-xanthylic acid using a microbial mutant strain is industrially beneficial. In this regard, the present inventors intended to develop a microbial mutant strain having remarkably enhanced productivity of 5'- guanylic acid by improving a character of conventional Corynebacterium aimoniagenes CJXFT0301 (KCCM-10530) to confer an ability to produce maximal levels of 5'-guanylic acid thereto. As a result, the present inventors found that a novel mutant strain, established by conferring sulfadiazine resistance to the known strain Corynebacterium ammoniagenes CJXFT0301 (KCCM-10530) , produces high concentrations and higher yields of 5'-xanthylic acid during the same fermentation duration by direct fermentation, thereby leading to the present invention.

Disclosure of the Invention

It is therefore an object of the present invention to provide a microorganism that has resistance to sulfadiazine and produces 5'-xanthylic acid in high concentrations and high yields by direct fermentation.

It is another object of the present invention to provide a method of producing 5'-xanthylic acid in high concentrations and high yields by culturing the microorganism. Best Mode for Carrying Out the Invention

The present invention relates to Corynebacterium ammoniagenes CJXSul0401 (KCCM-10609) that has sulfadiazine resistance and produces 5'-xanthylic acid in high concentrations and high yields.

In addition, the present invention provides a method of producing 5'-xanthylic acid by culturing Corynebacterium aimoniagenes CJXSul0401 (KCCM-10609) and directly accumulating 5'-xanthylic acid in high concentrations and high yields in culture fluid.

The present microorganism, Corynebacterium aimoniagenes CJXSul0401 (KCCM-10609) , is a mutant strain of Corynebacterium aimoniagenes CJXFT0301 (KCCM-10530) . Corynebacterium aimoniagenes CJXFT0301 (KCCM-10530), used as a parent strain in the present invention, is disclosed in Korean Pat. Application No. 10-2003-089714. The microorganism according to the present invention shares the following major characteristics and effectiveness of Corynebacterium aimoniagenes CJXFT0301 (KCCM-10530) . i) Requirement for adenine ii) Requirement for guanine iii) Susceptibility to lysozymes iv) Resistance to proline analogues v) Resistance to glutamine analogues vi) Resistance to tryptophan analogues In addition to the aforementioned properties, the present microorganism Corynebacterium ammoniagenes CJXSul0401 (KCCM-10609) has resistance to sulfadiazine and is thus capable of directly accumulating 5'-xanthylic acid in higher concentrations and higher yields in culture fluid.

The biosynthesis pathway of 5'-xanthylic acid (XMP) is very complicated and is a series of reactions in which various amino >acids and coenzymes are additionally involved. In particular, the XMP biosynthesis pathway consists of 11-stepwise reactions from phosphoribosylpyrophosphate (PRPP) to XMP. In the reactions, two molecules of glutamine, one molecule of glycine, one molecule of aspartate, one molecule of HCO₃- and two molecules of N₅N₁₀-methylenetetrahydrofolic acid (THF-CHO) are involved. Thus, increased addition of these materials involved in the formation of purine bases can help synthesize purine bases.

Sulfadiazine is a sulfa antibiotic that is a structural derivative of p-aminobenzoic acid (PABA) . Sulfadiazine interrupts the action of dihydropteroate synthetase, thereby blocking the production of an intermediate of folic acid synthesis, dihydrofolic acid. 'Due to this action, sulfadiazine inhibits the synthesis of tetrahydrofolic acid in microbes. Tetrahydrofolic acid plays critical roles in the synthesis of purine, thymidine, DNA, and the like. Thus, a decrease in tetrahydrofolic acid levels inhibits microbial growth. The sulfadiazine-resistant strain, CJXSul0401 (KCCM-10609) , is considered to increase the synthesis of dihydropteroate to overcome growth inhibition caused by sulfadiazine. This increases the amount of tetrahydrofolic acid essential for xanthylic acid synthesis. This increase in tetrahydrofolic acid levels leads to high-level synthesis of xanthylic acid, thereby making it possible to produce 5'-xanthylic acid in high concentrations and high yields by direct fermentation in comparison with conventional techniques. This finding leads to the present invention. A microorganism can be mutated by X-ray, ultraviolet light, chemical mutagens (e.g., N-methyl-N-nitro-N- nitrosoguanine, diethylsulfate, ethylamine, etc.), and the like. To obtain the microorganism according to the present invention, Corynebacterium ammoniagenes CJXFT0301 (KCCM- 10530) was used as a parent strain. The parent strain was treated with a mutagen, N-methyl-N-nitro-N-nitrosoguanine

(NTG) , according to a method commonly used in the art, and was screened on media supplemented , with varying concentrations of sulfadiazine, the media designated "M3" that is described in Table 2, in order to obtain mutant clones that can survive in the presence of sulfadiazine. The present microorganism was selected from the obtained mutant clones.

Sulfadiazine was contained in media used in tests of the present invention in an amount of 5 mg/L. The parent strain Corynebacterium ammoniagenes CJXFT0301 had resistance to sulfadiazine of up to 0.1 mg/L, and did not grow at 0.2 mg/L or higher concentrations of sulfadiazine. In contrast, the selected sulfadiazine-resistant strain, Corynebacterium ammoniagenes CJXSul0401 (KCCM-10609) , was found to grow even at 1 mg/L of sulfadiazine.

The selected mutant clone was designated

"Corynebacterium ammoniagenes CJXSul0401", which was deposited at the Korean Culture Center of Microorganisms

(KCCM; Hongjae 1-dong, Seodaemun-gu, Seoul, Republic of Korea) on November 16, 2004 under the provisions of the

Budapest Treaty and assigned accession number KCCM-10609.

The novel mutant strain CJXSul0401 (KCCM-10609), isolated in the present invention, has the biochemical properties described in Table 1 and is able to grow even in a medium supplemented with 1 mg/L of sulfadiazine.

The mutant strain of the present invention has sulfadiazine resistance properties described in Table 1, in which the mutant strain is compared with the parent strain CJXFT0301 for sulfadiazine resistance. TABLE 1

the strains were cultured at 30°C for 5 days.

A method of producing 5'-xanthylic acid using the novel strain of the present invention is as follows.

The Corynebacteriυm ammoniagenes CJXSul0401 (KCCM- 10609) strain according to the present invention is cultured in an ordinary medium containing proper carbon sources, nitrogen sources, amino acids and vitamins under aerobic conditions while factors such as temperate and pH are controlled.

Examples of available carbon sources include carbohydrates such as glucose, fructose and sterile pretreated molasses (i.e., molasses converted to reduced sugars) . Examples of available nitrogen sources include inorganic nitrogen sources, such as urea, ammonium chloride and ammonium sulfate, and organic nitrogen sources, such as peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydrolysates, fish or decomposition products thereof, and defatted soybean cake or decomposition products thereof.

Examples of available inorganic compounds include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate and calcium carbonate. The culture medium may be further supplemented with vitamins, auxotrophic bases, and the like, according to the intended use. Cultivation is carried out under aerobic conditions, for example, with shaking or aeration-agitation, at preferably 20 to 40°C. Culture media are preferably maintained near neutral pH during cultivation. Cultivation lasts for 5 to 6 days. 5'-xanthylic acid, accumulated by direct fermentation, is analyzed according to an ordinary method.

As a result, when 5'-xanthylic acid was produced by direct fermentation using the parent strain CJXFT0301, 152.2 g/L of 5'-xanthylic acid was obtained. In contrast, when the present mutant strain, Corynebacterium aimoniagenes CJXSul0401, was used, 160.4 g/L of 5'- xanthylic acid was obtained under the same conditions. These results indicate that the CJXSul0401 strain, acquired sulfadiazine resistance, has improved productivity of 5¹- xanthylic acid in comparison with the parent strain CJXFT0301.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention. EXAMPLES

Microbial cultivation in the present invention was conducted using culture media described in Table 2, below, including a nutrient medium (designated "M1") , a minimal medium (designated "M2") , a sulfadiazine-containing medium (designated "M3") , a medium for seed culture in a flask (designated "M4") , a medium for main fermentation in a flask (designated "M5") , a separately autoclaved medium for flask fermentation (designated "M6") , a medium for first- seed culture in a fermentor (designated "M7") , a medium for second-seed culture in a fermentor (designated "M8") , and a medium for main fermentation in a fermentor (designated "M9") . However, culture media are not limited to the compositions listed in Table 2, and all culture media suitable for cultivation of Corynebacterium ammoniagenes are available.

TABLE 2

EXAMPLE 1 : Establishment of sulfadiazine-resistant strain

A mutant strain according to the present invention was established as follows . Corynebacterium ammonia genes CJXFT0301 (KCCM-10530) was used as a parent strain and treated with N-methyl-N-nitro-N-nitrosoguanine at a final concentration of 30 μg/ml at 30 °C for 60 min in order to induce mutation. Then, the parent strain was washed with 0.85% physiological saline three times, suitably diluted with a medium prepared by adding 1 mg/L of sulfadiazine to a minimal medium (M2 ) containing 1.7% agarose, and smeared on a solid medium. Each of the emerged colonies was cultured in a nutrient medium (Ml ) , a seed culture medium

(M4 ) for 24 hrs, and then a mixed broth of a fermentation medium (M5) and a separately autoclaved medium (M6) for 4 days . Of the colonies, a clone having the highest productivity of 5 ' -xanthylic acid, which was accumulated in culture fluid, was selected. The selected clone was designated "Corynebacterium ammoniagenes CJXSul0401", which was deposited at the Korean Culture Center of Microorganisms (KCCM; Hongjae 1-dong, Seodaemun-gu, Seoul, Republic of Korea) on November 16, 2004 under the provisions of the Budapest Treaty and assigned accession number KCCM-10609.

EXAMPLE 2 : Fermentation titer test in Erlenmeyer flasks

Strains used: CJXSul0401 and CJXFT0301 Medium for seed culture in flask: M4 listed in Table 2 Medium for main fermentation in flask: M5 listed in Table 2 separately autoclaved medium for flask fermentation : M6 listed in Table 2

5 ml of the seed culture medium was aliquotted into test tubes 18 mm in diameter and autoclaved under pressure according to an ordinary method. Each of the strains was inoculated in the sterile seed culture and incubated at 30 °C for 18 hrs with shaking at 180 rpm. This seed culture was used as an inoculum for fermentation. The medium for main fermentation and the separately autoclaved medium were individually autoclaved under pressure according to an ordinary method, and were aliquotted at 29 ml and 10 ml, respectively, into 500-ml shaking Erlenmeyer flasks . 1 ml of the seed culture was inoculated in the flasks and incubated for 90 hrs . The incubation was carried out at 30°C with shaking at 200 rpm. Thereafter, the amount of 5 ' - xanthylic acid thus produced and accumulated in culture fluid was determined. 5 ' -xanthylic acid was found to be accumulated in culture fluid at 28. 6 g/L by the conventional strain CJXFT0301 (KCCM-10530) and 30.7 g/L by the present mutant strain CJXSul0401 (KCCM-I 0609) . The accumulated concentration of 5 ' -xanthylic acid (XMP) is given by 5 ' -XMP • Na₃ • 7H₂O.

EXAMPLE 3 : Fermentation titer test in fermentors

Strains used: CJXSul0401 and CJXFT0301 Medium for first-seed culture in fermentor: M7 listed in Table 2

Medium for second-seed culture in fermentor : M8 listed in Table 2 Medium for main fermentation in fermentor : M9 listed in Table 2

First-seed culture: 50 ml of the first-seed culture medium was aliquotted into a 500-ml shaking Erlenmeyer flask, autoclaved at 121°C for 20 min under pressure, and allowed to cool. Each strain was inoculated in the sterile culture medium and incubated at 30°C for 24 hrs with shaking at 180 rpm. Second-seed culture: 2 liters of the second-seed culture medium was aliquotted into a 5-L experimental fermentor, autoclaved at 121°C for 20 min under pressure, and allowed to cool. 50 ml of the first-seed culture was inoculated in the fermentor and incubated at 31°C for 24 hrs with agitation at 900 rpm and a aeration of 0.5 wm. The medium was adjusted during fermentation to pH 7.3 with aqueous ammonia.

Fermentation: 8 liters of the medium for main fermentation was aliquotted into a 30-L experimental fermentor, autoclaved at 121°C for 20 min under pressure, and allowed to cool. 1.5 L of the second-seed culture was inoculated in the fermentor and incubated at 33°C with agitation at 400 rpm and a aeration of 1 wm. During fermentation, sterile glucose was supplied to the medium when residual sugar levels were less than 1% in order to adjust total sugar in the medium for main fermentation to 30%. The pH of the medium was adjusted during fermentation to 7.3 with ammonia gas. The fermentation was conducted for 80 hrs. Thereafter, the amount of 5'-xanthylic acid thus produced and accumulated in the medium was determined. 5'- xanthylic acid was found to be accumulated in the medium at 152.2 g/L by the conventional strain CJXFT0301 (KCCM-10530) and 160.4 g/L by the present mutant strain CJXSul0401 (KCCM-10609) . The accumulated concentration of 5'-xanthylic acid (XMP) is given by 5'-XMP • Na₃ • 7H₂O.

Industrial Applicability

As described hereinbefore, the strain Corynebacterium armoniagenes CJXSul0401 (KCCM-10609) according to the present invention is capable of economically producing 5'- xanthylic acid in higher concentrations and higher yields by direct fermentation than a conventional strain.

Claims

1. A microorganism Corynebacterium aimoniagenes CJXSul0401 having accession number KCCM-10609, the microorganism having sulfadiazine resistance and producing 5'-xanthylic acid.

2. A method of producing 5'-xanthylic acid, which is based on culturing the microorganism according to claim 1 to produce 5'-xanthylic acid in culture fluid.